The organization and asymmetry inherent in cell membranes creates an environment in which receptor proteins can effectively convey information between the inside and outside of the cell (1). The reduction in the degrees of freedom experienced by transmembrane and peripheral membrane proteins provides a strong driving force for lateral organization, which can be essential for function, e.g. ligand-induced clustering (2). These factors are in effect at the plasma membrane inner leaflet, where the assembly and regulation of signaling components often occur (3-5). In the chemotaxis signal transduction pathway of Escherichia coli, complexes of transmembrane receptors and cytoplasmic signaling proteins (6,7) regulate protein phosphorylation through ligand-receptor interactions and receptor covalent modification (8-12). This transduction pathway is coupled to cell motility, which biases the swimming behavior of the cell in attractant and repellant gradients (13).
Several lines of evidence suggest that receptors are clustered in the cell membrane (14-16) and that close associations between receptors of different ligand specificity are important in signaling (17-21). The aspartate receptor (Tar) is representative of a large class of receptors in the bacterial chemotaxis pathways (23,24), which are also known as the methyl-accepting chemotaxis proteins (MCPs) due to the enzyme-catalyzed receptor methylation and demethylation reactions that are essential for sensory adaptation (25). The structure of the Tar dimer (FIG. 1A) provides insights into the basis for the possible requirement of receptor clusters in signaling. Escherichia coli has four MCPs (Tar, Tsr, Tap, Trg) and an aerotaxis receptor (Aer) that are distinguished by ligand binding specificities, which reside primarily in the n-terminal extra-cytoplasmic domain. The dimeric organization of MCPs is evident in crystal structures of the aspartate receptor (Tar) ligand binding domain (26,27) and the serine receptor (Tsr) cytoplasmic domain (28). The significantly greater homology among the c-terminal domains of these five receptors provides the basis for common interactions among a set of cytoplasmic signaling proteins (23), which generate the excitatory and adaptive responses to the chemotactic stimuli (reviewed in reference 29). In addition, the Tsr cytoplasmic domain is organized as a trimer-of-dimers in the crystal structure (28). The subunit interactions that lead to the trimer-of-dimer structure are apparently important for the intact receptor in the cell, since mutations in conserved amino acid residues at the trimer-of-dimer contact site disrupt chemotaxis and receptor clustering (21). Thus, within the context of these heterogeneous receptor clusters, the overall signaling protein (e.g., CheA in E. coli) activity reflects the influences of the various independent inputs, detected by the MCPs and Aer.
Biochemical investigations using membrane preparations of either Tar or Tsr with the purified signaling proteins have clarified some of the properties of CheA activation and regulation (8-12). Notable observations include the substantial increase in CheA activity (>100-fold) that accompanies signaling complex formation, the stimulating influence of receptor methylation on CheA activity, and the inhibitory influence of ligand binding. However, membrane samples of the MCPs that are used in such biochemical experiments are frequently isolated from cells expressing the receptor at elevated levels, which can result in complex and heterogeneous samples (30). Also, receptor reconstitution is labor-intensive, and the conditions that maintain a high level of activity while also preserving the vectoral and lateral organization required for function can be difficult to find (31,32). For example receptor organization leads to purification difficulties involving procedures that invariably require two-phase detergent containing systems. Low yields are typical and represent an impediment to widespread use of such receptors in cell-free assay systems. An added disadvantage to the use of a homogeneous assay is the detergent, itself, which disrupts the interactions between receptor proteins on a membrane.
To circumvent the difficulties that typically plague the use of such samples, studies of CheA activation have used soluble cytoplasmic receptor fragments (CFs). In many instances CFs are unable to activate CheA, but those that do seem to via oligomerization, which occurs synergistically with CheW and CheA binding (33-36). While this approach has helped to elucidate the enzymatic properties of signaling complexes, the formation of these complexes is limited to certain relative concentrations of CF, CheW and CheA, and is undesirably sensitive to variations in the tendency of different CFs to oligomerize. As a result, a comprehensive study of the factors important for CheA activation remains a continuing research goal.
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